This invention relates generally to methods for removing sodium dodecyl sulfate (SDS) from a solution and particularly to a method using guanidine hydrochloride (GCl) to remove excess SDS from SDS-solubilized protein solutions.
Heterologous DNA segments that encode for a particular protein can be inserted into host microorganisms using recombinant DNA technology. By growing the transformant microorganisms under conditions which induce the expression of proteins, heterologous proteins such as insulin, somatotropins, interleukins, interferons, somatomedins, and the like can be produced in large quantities at a relatively low cost.
Unfortunately, heterologous proteins produced by transformant microorganisms are frequently not biologically active because they do not fold into the proper tertiary structure when transcribed within the microorganism. The heterologous proteins tend to form aggregates which are recognizable within the cell as "inclusion bodies". These inclusion bodies may also be caused by the formation of covalent intermolecular disulfide bonds which link together several protein molecules to form insoluble complexes. The inclusion bodies generally contain mostly heterologous protein and a small fraction of contaminating host microorganism proteins.
Several processes have been developed to extract the inclusion bodies from the microorganisms and convert the heterologous proteins contained therein into proteins having native bioactivity consistent with the natural parent or non-recombinant proteins. These processes generally involve disrupting the microorganism cell, separating the inclusion bodies from cell debris, solubilizing the inclusion body proteins in a denaturant/detergent which unfolds the protein, separating the heterologous inclusion body proteins from contaminating proteins, and removing the denaturant/detergent thereby allowing the heterologous proteins to refold into a bioactive tertiary conformation. These general steps may be carried out in different orders and using several different techniques, equipment, and chemicals.
Several purification schemes following this general outline have been developed: U.S. Pat. No. 4,511,502 discloses a process wherein the solubilized protein solution is passed over a molecular sieve or centrifuged to remove high molecular weight contaminating proteins. The denaturant is subsequently removed by dialysis to allow the protein to refold into its bioactive conformation. U.S. Pat. No. 4,511,503 discloses solubilizing inclusion body proteins with a strong denaturant. The strong denaturant permits the improperly folded protein molecules to unfold and become soluble in the denaturant solution. The denaturant is subsequently removed by dialysis to allow the protein to refold into a bioactive conformation.
U.S. Pat. No. 4,518,526 discloses a process wherein transformed cells are treated with a buffered solution of sufficient ionic strength to solubilize most of the whole cell protein while leaving the heterologous protein in insoluble aggregates. The transformed cells are lysed and the supernatant containing the solubilized whole cell proteins is separated from the insoluble inclusion bodies. The inclusion bodies are then solubilized using a strong denaturant.
Each of these patents deals extensively with the use of concentrated guanidine hydrochloride (GCl) as a denaturant and suggests the use of SDS as a detergent/denaturant in the process. There are, however, no methods disclosed for using SDS in recovery procedures nor for removing SDS from the protein solution to allow refolding. Presumably, SDS could be removed using dialysis and other techniques disclosed in the patent for the removal of GCl, although this is rendered less practical because of the low critical micelle concentration and large micellar size of the detergent.
Unfortunately, these techniques are often incompatible with current protein purification procedures. Also, the reagents and process conditions used during purification often induce protein reaggregation and precipitation, thus reducing the yield and increasing production costs.
Another method for recovering the heterologous protein in bioactive form comprises separating inclusion bodies from cell debris solubilizing the inclusion bodies in SDS, separating the SDS-heterologous protein complexes from those containing contaminating proteins, and removing the SDS from the heterologous protein solution using chromatography. The SDS must be added in sufficient amounts to form SDS-protein complexes. Typically an excess of SDS is added to insure complete protein solubilization. This excess, non-protein-bound SDS remains in the solution and must be removed before the protein can be restored to its bioactive conformation. As the SDS is removed the protein refolds into its bioactive tertiary structure.
Purification schemes using SDS as a denaturant/detergent generally involve solubilizing the inclusion body by adding excess SDS to denature/unfold the proteins. SDS exists in solution in two forms: (1) SDS bound to the protein in a SDS:protein complex and (2) excess, non-protein-bound SDS in solution. SDS is removed by dialysis, ion retardation chromatography, or other suitable means to allow the protein to refold into a bioactive conformation. The resulting protein is bioactive at this stage or, if not bioactive at this stage, can be further processed to produce a purified bioactive protein.
Several methods are available for the removal of SDS. Kapp et al., Anal. Biochem., 91:230-33(1978) discloses a method for removing SDS from SDS-protein solutions using ion-retardation chromatography resin AG11A8. The problem with this method is the time required to chromatograph large quantities of SDS-proten solution and the cost involved with regenerating or replacing the ion-retardation columns. The non-protein-bound SDS overloads the column and requires frequent column regeneration and replacement. The non-bound SDS could possibly be removed by "buffer exchange" or dialysis techniques but this would involve extra time-consuming and expensive steps. Weber et al., J. Biol. Chem., 246:4504-09(1971) demonstrated that SDS could be removed from aspartate transcarbamoylase by incubation in urea followed by anion-exchange chromatography.
Various methods for the removal of SDS from SDS-protein solutions, whether by dialysis, anion-exchange chromatography, ion-retardation chromatography, or other suitable means, share a common problem. The excess, non-protein-bound SDS must be removed before the protein-bound SDS can be removed. If dialysis is used to remove the excess SDS, large quantities of buffer must be dialyzed against the SDS-protein solution for long periods of time to remove the SDS. If chromatography is used, the SDS often saturates the column requiring frequent column replacement or regeneration. These problems are caused by the non-protein-bound SDS which was added in excess to insure the complete solubilization of the inclusion bodies. Less frequent column replacement or regeneration would be required and more optimal throughput of protein-containing feed solutions would result if a method were to exist for removing free or excess SDS before dissociating the bound SDS. A method is, therefore, needed for quickly and efficiently removing excess SDS from SDS-protein solutions. Such a method forms the subject of this invention.